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Quinine sulfate and HSV replication: Implications in malaria-endemic areas

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Quinine sulfate and HSV replication
Ronni Wolf1, Adone Baroni2, Rita Greco3, Federica Corrado3, Eleonora Ruocco2, Maria Antonietta Tufano3, and Vincenzo Ruocco2
Dermatology Online Journal 9(3): 3

1. Dermatology Unit, Kaplan Medical Center, Rechovot, 2. Department of Dermatology, Faculty of Medicine and Surgery, Second University of Naples 3. Department of Experimental Medicine, Microbiology Section, Faculty of Medicine and Surgery, Second University of Naples. MariaAn.tufano@unina2.it

Abstract

Although antimalarial drugs have been developed primarily to treat malaria, they are also beneficial for many dermatological, immunological, and rheumatological diseases, for which they are mostly used today in the Western world. The aim of the present study was to investigate the effect of quinine sulfate (QS) on the multiplication and adsorption of herpes virus type I (HSV-1). When Vero cells (African green monkey kidney) are infected with HSV-1 in the presence of QS, the viral adsorption is reduced, as demonstrated by a decrease of the number of microscopic plaques of the virus. When the virus-infected Vero cells are incubated in the presence of QS, the multiplication of HSV-1 is also reduced, and the diameter of the plaque are visibly smaller. The practical implications of the antiviral action of antimalarial drugs might be especially important to immunosuppressed patients who receive these drugs for autoimmune collagen-vascular diseases or as additional therapy for AIDS.



Introduction

Antimalarial drugs were first discovered in the seventeenth century, two and one-half centuries before the causative agent of malaria was identified.[1] Quinine, the first antimalarial agent, was derived from the cinchona tree. Quinine played a unique role in shaping today's world by enabling explorers and colonists from Europe to survive in tropical countries and to build their colonial empires despite the presence of lethal tropical malaria. When physicians were asked in 1945 to choose the ten most important drugs used in medicine, quinine and quinacrine were preceded in importance only by penicillin, sulfonamides, and blood derivatives.[2] Although antimalarial drugs have been developed primarily to treat malaria (and have never lost their place in treating this life-threatening disease that is still a major cause of infant death in the tropics), they are also beneficial for many dermatological, immunological, and rheumatological diseases, conditions for which they are used today mostly in the Western world.[3, 4]

In the beginning of the 1970s, researchers became interested in another property of antimalarial drugs, their antiviral activity. Thus ensued an evaluation of the affect of these substances on viruses of various structures and modes of replication. [5, 6, 7, 8, 9, 10, 11, 12, 13, 14]

The search for viruses that could be affected by antimalarials has continued over the years, and has resulted in an ever-growing list in which replication, activity, or infectivity are inhibited by these drugs. Antimalarials are found to affect vesicular-stomatitis virus[15, 16, 17], foot-and-mouth disease[18], yellow fever virus[19], varicella-zoster virus[20], Newcastle disease virus[21], African swine fever virus[22], Flavivirus[23], canine parvovirus[24], Epstein-Barr virus (EBV)[25], lymphocytic choriomeningitis virus[26, 27], Semliki Forest virus[28], bovine leukemia virus[29], feline calicivirus[30], infectious salmon anemia virus[31], rubella virus[32], hepatitis A virus[33], hepatitis B virus[34, 35, 36], rabies virus[37], Ebola virus[38], Borna disease virus[39], and porcine reproductive and respiratory syndrome virus.[40, 41] Some other viruses, such as poliovirus, are unaffected by antimalarials.[42, 43, 44]

The effect of antimalarials on herpes virus (HSV) is among the first to be studied. In 1971, Greenham and Poste show that lysosomal activation is a consistent feature of cell fusion induced by a range of viruses, including HSV.[10, 11] Lysosome-stabilizing agents (among them chloroquine) significantly reduce cell fusion without impairing virus replication. In contrast, Lancz et al. find that HSV replication is inhibited by chloroquine.[45] The higher concentrations of chloroquine in their experiment (using 60 µg/ml compared to 30 µg/ml in the earlier experiment) might be one explanation for the difference in the results. The findings of Lancz et al. are confirmed 2 years later by other authors, who also perform an in vivo experiment inoculating mice with HSV-1 and HSV-2 and find the course of infection to be unaltered by chloroquine treatment.[46]

Other investigators are able to demonstrate that 100 µM chloroquine inhibits multiplication of HSV-1 cultured in Vero cells in the late stage of infection, a result that indicates that the site of inhibition is in one of the steps within the maturation process of the virus.[47, 48] This inhibition is not attributed to any interference with the uncoating process. The drug also inhibits the syncytium formation induced by HSV-1 but does not affect the cell-to-cell spread of infection.

In the present study we investigate the effect of quinine sulfate (QS) on HSV-1 multiplication and adsorption.


Materials and methods


Cells and culture conditions

Vero cells (African green monkey kidney) were grown in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10-percent heat-inactivated fetal calf serum (FCS), in flask 25 cm2 at 37° C in a 5-percent CO2 incubator. Cells were grown to confluence for 3 days.


Chemicals

QS (molecular weight 783 kDa) was dissolved in a 1:1 mixture of ethanol and water.


Toxicity of QS on cultured cells

The cells that had propagated in tissue-culture clusters were incubated with different concentrations of QS (0, 1, 10, 25, 50, and 100 µM) at 37° C for 24, 48, and 72 hours in RPMI medium. The cell monolayers were then washed with phosphate-buffered saline (PBS) and stained with Giemsa diluted 1:20.


Virus preparation and infections

HSV-1 was propagated in vitro by infecting Vero cells at a multiplicity of infection (MOI) of 10.

Cells were incubated in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 percent FCS. At about 3 days post-infection, the virus was harvested by sonication and clarified by centrifugation.

A virus stock solution containing approximately 109 plaque-forming units (PFU)/ml was used in all experiments.

Monolayer Vero cells were cultured in a multiwell plate containing 12 wells and washed with Dulbecco's PBS. Then 0.2 ml virus dilutions with or without QS were added at concentrations of 1 µM and 10 µM, at a MOI of 0.1 and 0.01. After incubating the multiwell plate for 1 hour at 37° C, the inocula were removed, and the infected monolayers were incubated for additional 72 hours in RPMI medium with carboxymethyl cellulose (CMC) (ratio1:1) with or without QS.

The cells were fixed and stained with 1 percent crystal violet dissolved in a 10 percent formalin solution, and the number of microscopic plaques was counted under a microscope.


Results

QS is cytocidal at concentrations of 25, 50, and 100 µM, but not at concentrations of 1 µM and 10 µM. The cells that are incubated for 24, 48, and 72 hours at these lower concentrations are viable. Moreover, QS at concentrations of 1 µM and 10 µM does not show any virucidal activity.

When the cells are infected with HSV-1 in the presence of QS, the viral adsorption is reduced as demonstrated by a decrease of the number of microscopic plaques of the virus, expressed as PFUs (Table I).

Table 1
Effect of quinine sulfate (QS) on adsorption and replication
of herpes simplex virus type 1 (HSV-1) in Vero cells
Condition of
incubation with QS
MOIHSV-1HSV-1
with QS
1mM
% of
decrease
of PFU
HSV-1
with QS
10 mM
% of
decrease
of PFU
Preadsorption0.17804324510886
Pre- and
postadsorption
0.17003205419272.5
Preadsorption0.01476128739081
Pre- and
postadsorption
0.0143252884090
Results are expressed as plaques forming units (PFU)/ml.
MOI = multiplicity of infection.

When the virus-infected Vero cells are incubated in the presence of QS the multiplication of HSV-1 is also reduced and the diameters of the plaques are visibly smaller (Fig. 1).


Figure 1

Discussion

We are able to show that QS reduces adsorption and multiplication of HSV-1 at very low concentrations (1 µM and 10 µM), levels more than ten-fold lower than the 50-percent inhibitory concentration of the drug (IC50), as well as lower than concentrations of chloroquine used on viruses in previously reported experiments by others.[49, 50, 51]

Apart from the theoretical considerations of our findings, they might also have practical implications in illuminating the antiviral action of antimalarial drugs, especially in regard to immunosupressed patients who receive these drugs for autoimmune-collage-vascular diseases, or as additional therapy for AIDS.

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